The present invention provides methods and compositions for the in situ growth, freezing and testing of cultured cells. In particular, the present invention provides methods and compositions for the long-term preservation of cells in ready-to-use formats for testing. In addition, the present invention provides rapid and easy to use means to diagnose viral and other infections. Furthermore, the present invention provides easy to use means to grow and store cells in situ for testing methods. Indeed, the present invention makes viral, chlamydial and other diagnostic methods accessible to small laboratories, including those without cell culture capabilities.
As intracellular parasites (e.g., viruses and Chlamydia) require living cells in order to replicate, diagnosis of infection due to these organisms relies upon the use of either animals (e.g., suckling mice), embryonated eggs, or cell cultures. As cell cultures are much less expensive and are easier to work with than animals or embryonated eggs, cell cultures have long been the mainstay of diagnosis methods for intracellular parasites and viruses in particular. Indeed, cell cultures are the foundation upon which a virology laboratory is built. These cultures may be produced in house from animal tissues or organs, or more commonly, purchased from commercial suppliers.
Regardless of their sources, cell cultures must be maintained over time, in order to ensure a ready supply of cells for growth and diagnosis of infections caused by intracellular parasites. In the laboratory, mammalian cells are routinely frozen in order to minimize the opportunity for contamination of the cultures, guard against handling errors that could result in the loss of the culture, and minimize the number of cell lines that must be handled on a daily basis. Frozen cell culture stocks are also useful for minimizing genetic drift and shift, senescence, and undesirable phenotypic changes that can occur when continuous and finite cell lines are cultured for long time periods.
Freezing methods have been developed to minimize the impact of osmotic shock and intracellular ice crystal formation, two factors that contribute to the loss of cell viability during freezing. Cryoprotectants such as glycerol and dimethylsulfoxide (DMSO) are commonly used to help prevent cell death during freezing. In addition to the use of cryoprotectants, traditional methods use slow cooling (approximately 1xc2x0 C. per minute) until the cells reach a temperature of xe2x88x9225xc2x0 C. Once this temperature is attained, the cells can be rapidly cooled to xe2x88x9270xc2x0 C. or xe2x88x92196xc2x0 C. (i.e., liquid nitrogen temperature), without further loss of cell viability. Omitting the cryoprotectant or rapid freezing causes the formation of intracellular ice crystals which can rupture cell membranes and result in cell death. By slowly cooling the cells, the external medium becomes supercooled and ice crystal nuclei form in the extracellular fluid. This results in an extracellular environment that contains an artificially elevated salt gradient which, in turn, causes an osmotic gradient. This gradient causes water to diffuse out of the cells and the nonelectrolyte cryoprotectants to diffuse into the cells. This xe2x80x9cdehydrationxe2x80x9d of the cells tends to minimize osmotic shock and intracellular ice crystal formation. (See e.g., Wiedbrauk and Johnston, xe2x80x9cMammalian Cell Culture Procedures, in Manual of Clinical Virology, pages 33-44 [Raven Press, New York, 1993], for a description of these events).
However, commonly used freezing methods require specialized equipment and training. In addition, hazardous chemicals such as DMSO are typically used. Furthermore, thawing of frozen cells maintained in liquid nitrogen poses risks such as explosion of the vials or tubes as well as the danger of loss of cell viability due to improper handling (e.g., slow, rather than rapid thawing). Once the cells have been thawed, the freezing medium must be removed and rinsed from the cells and the culture revived prior to use for growth and/or detection of intracellular parasites. Once revived, the cultures are often placed into formats suitable for the detection and identification of viruses, including multiwell plates (e.g., microtiter plates), tubes, and slides). Thus, the cultures must be transferred from their growth flask to these other formats prior to their use. This necessitates additional equipment and personnel time, prior to the use of the cultures as desired. What is needed are cell cultures and methods that are easy to use, readily available, particularly in ready-to-use formats, require little operator time and/or experience to use, and are reliable.
The present invention provides methods and compositions for the in situ growth, freezing and testing of cultured cells. In particular, the present invention provides methods and compositions for the long-term preservation of cells in ready-to-use formats for testing. In addition, the present invention provides rapid and easy to use means to diagnose viral and other infections. Furthermore, the present invention provides easy to use means to grow and store cells in situ for testing methods. Indeed, the present invention makes viral, chlamydial and other diagnostic methods accessible to small laboratories, including those without cell culture capabilities.
The present invention provides methods for the detection of intracellular parasites in a sample, comprising the steps of providing a cell culture comprising cells, wherein the cell culture has been frozen and thawed in situ on a substrate, and a sample suspected of containing at least one intracellular parasite; adding the sample to the cell culture to produce an inoculated culture; incubating the inoculated culture under conditions such that the intracellular parasite infects the cells of the cell culture to produce an infected culture; and observing the infected culture for the presence of the intracellular parasite within the cells of the cell culture. In some embodiments, the substrate is selected from the group consisting of glass and plastic. In some preferred methods, the glass is a glass coverslip. In still other preferred embodiments, the plastic substrate is the well of a multiwell plate. In some embodiments, the intracellular parasite is selected from the group consisting of viruses and bacteria.
In some embodiments, the observing comprises observing for the presence of cytopathic effect, while in other embodiments, the observing comprises observing for the presence of fluorescent cells. In still other embodiments, the observing comprises observing for the presence of blue cells. In some preferred embodiments, multiple observations are made. For example, in some embodiments, observing for cytopathic effect is combined with observing for fluorescent cells, and/or observing for blue cells. As described herein, in preferred embodiments, the observing for fluorescent cells is accomplished using labeled antibodies that recognize an antigen (e.g., viral or bacterial) present in the culture (i.e., due to the infection of the cells by the virus(es) and/or bacteria). However, it is not intended that the present invention be limited to any particular fluorescence product, substrate, enzyme, or color. Indeed, it is intended that multiple antibodies and fluorescence labels will find use in some embodiments of the present invention. For example, it is contemplated that the cells of the present invention will be tested using multiple antibody preparations with differing fluorescent labels. In addition, it is not intended that the present invention be limited to fluorescently labeled antibodies, as other detection means will find use with the present invention. Also as described herein, the observing for blue cells is associated with test methods such as those that involve the use of a reporter gene which indicates that a particular gene is being expressed. However, it is not intended that the present invention be limited to any particular gene, product, substrate or color. Indeed, it is contemplated that the present invention will find use with multiple genes. products, substrates, and/or colors (i.e., multiple reporter genes will be used). Furthermore, in some particularly preferred embodiments, the use of fluorescent label(s) is combined with enzymatic method(s).
The present invention also provides methods for the detection of a toxin in a sample, comprising the steps of providing a cell culture comprising cells, wherein the cell culture has been frozen and thawed in situ on a substrate, and a sample suspected of containing at least one toxin; adding the sample to the cell culture to produce an inoculated culture; incubating the inoculated culture to produce an intoxicated culture; observing the intoxicated culture for the presence of cytopathic effect on the cells of the cell culture. In some embodiments, the substrate is selected from the group consisting of glass and plastic. In some preferred methods, the glass is a glass coverslip. In still other preferred embodiments, the plastic substrate is the well of a multiwell plate. In some preferred embodiments, the toxin is a Clostridium toxin, while in particularly preferred embodiments, the toxin is a Clostridium difficile toxin. However, it is not intended that the present invention be limited to Clostridium toxin. Indeed, it is intended that the present invention encompass the detection of various other toxins, including but not limited to verotoxins, mycotoxins, and other toxins of interest, in particular those that cause disease and/or pathological changes in a host upon exposure to the toxin. In addition, it is contemplated that the present invention will find use in detecting and/or identifying multiple toxins present in a single sample. It is also intended that the toxin(s) will be identified and/or detected using methods other than the observation and characterization of cytopathic effect. Thus, it is intended that the present invention encompasses the detection and/or identification of toxins using any suitable method, including but not limited to the use of antibodies, toxin substrate analogs, reporter genes, etc.
The present invention also provides methods for the production of frozen ready to use cell cultures for in situ diagnostic assays, comprising the steps of providing cells, and a substrate selected from the group consisting of glass and plastic; placing the cells on the substrate under conditions such that the cells are attached to the substrate to produce a cell monolayer; and freezing the cell monolayer under conditions such that the cell monolayer remains attached to the substrate. In some preferred embodiments the substrate is plastic, while in some particularly preferred embodiments, the plastic comprises the well of a multiwell plate. In still further preferred embodiments, the substrate is glass. In other preferred embodiments, the glass is a glass coverslip, and in particularly preferred embodiments, the glass coverslip is placed in a shell vial prior to freezing of the monolayer. The present invention also provides compositions comprising a cell monolayer produced according to these methods.
The present invention also provides methods for the detection of intracellular parasites in a sample, comprising the steps of providing the ready-to-use cell monolayer produced as described above, and sample suspected of containing at least one intracellular parasite; thawing the cell monolayer; adding the sample to the cell monolayer to produce an inoculated culture; incubating the inoculated culture under conditions such that the intracellular parasite infects the cells of the cell monolayer to produce an infected culture; and observing the infected culture for the presence of the intracellular parasite within the cells of the cell monolayer. In some embodiments, the intracellular parasite is selected from the group consisting of viruses and bacteria.
In some embodiments, the observing comprises observing for the presence of cytopathic effect, while in other embodiments, the observing comprises observing for the presence of fluorescent cells. In still other embodiments, the observing comprises observing for the presence of blue cells. In some preferred embodiments, multiple observations are made. For example, in some embodiments, observing for cytopathic effect is combined with observing for fluorescent cells, and/or observing for blue cells. As described herein, in preferred embodiments, the observing for fluorescent cells is accomplished using labeled antibodies that recognize an antigen (e.g., viral or bacterial) present in the culture (i.e., due to the infection of the cells by the virus(es) and/or bacteria). However, it is not intended that the present invention be limited to any particular fluorescence product, substrate, enzyme, or color. Indeed, it is intended that multiple antibodies and fluorescence labels will find use in some embodiments of the present invention. For example, it is contemplated that the cells of the present invention will be tested using multiple antibody preparations with differing fluorescent labels. In addition, it is not intended that the present invention be limited to fluorescently labeled antibodies, as other detection means will find use with the present invention. Also as described herein, the observing for blue cells is associated with test methods such as those that involve the use of a reporter gene which indicates that a particular gene is being expressed. However, it is not intended that the present invention be limited to any particular gene, product, substrate or color. Indeed, it is contemplated that the present invention will find use with multiple genes, products, substrates, and/or colors (i.e., multiple reporter genes will be used). Furthermore, in some particularly preferred embodiments, the use of fluorescent label(s) is combined with enzymatic method(s).
The present invention also provides methods for the detection of toxin in a sample, comprising the steps of providing a ready-to-use cell monolayer produced as described above, and a sample suspected of containing at least one toxin; adding the sample to the cell monolayer to produce an inoculated culture; incubating the inoculated culture to produce an intoxicated culture; and observing the intoxicated culture for the presence of cytopathic effect on the cells of the cell monolayer. In some embodiments, the toxin is a Clostridium toxin, while in particularly preferred embodiments, the toxin is a Clostridium difficile toxin. However, it is not intended that the present invention be limited to Clostridium toxin. Indeed, it is intended that the present invention encompass the detection of various other toxins, including but not limited to verotoxins, mycotoxins, and other toxins of interest, in particular those that cause disease and/or pathological changes in a host. In addition, it is contemplated that the present invention will find use in detecting and/or identifying multiple toxins present in a single sample. It is also intended that the toxin(s) will be identified and/or detected using methods other than the observation and characterization of cytopathic effect. Thus, it is intended that the present invention encompass the detection and/or identification of toxins using any suitable method, including but not limited to the use of antibodies, toxin substrate analogs, reporter genes, etc.
The present invention also provides compositions comprising frozen, ready-to-use cell culture suitable for the detection of pathogenic substances, wherein the pathogenic substances are selected from the group consisting intracellular parasites and toxins. In some embodiments, the intracellular parasites are selected from the group consisting of viruses and bacteria. In particularly preferred embodiments, the bacteria are members of the genus Chlamydia. In other embodiments, the toxin(s) comprises at least one Clostridium toxin, while in other preferred embodiments, the toxin is a C. difficile toxin.
The present invention provides methods and compositions for the in situ growth, freezing and testing of cultured cells. In particular, the present invention provides methods and compositions for the long-term preservation of cells in ready-to-use formats for testing. In addition, the present invention provides rapid and easy to use means to diagnose viral and other infections. Furthermore, the present invention provides easy to use means to grow and store cells in situ for testing methods. Indeed, the present invention makes viral, chlamydial and other diagnostic methods accessible to small laboratories, including those without cell culture capabilities.
The cell cultures of the present invention are frozen in a format that is ready for testing upon thawing of the cells. In particularly preferred embodiments, the cells are frozen on coverslips placed within vials (i.e., shell vials). In these embodiments, the cells are frozen on a glass substrate without the need for pre-starvation or any special handling of the cells prior to freezing. In addition, the cells do not require any special handling during thawing or use. Thus, the preparation of the ready-to-use cell cultures can be accomplished in an economical and cost-effective manner. This is unlike the methods currently available that require a starvation step and/or lengthy recovery of the cells following freezing (See e.g., U.S. Pat. No. 5,935,855, herein incorporated by reference). Thus, the present invention provides cells in a ready-to-use format that requires minimal handling both prior to the freezing and after the thawing of the monolayers. In addition to the preferred shell vial embodiments in which the cells are attached to a coverslip, the present invention provides cell monolayers attached to plastics (e.g., the plastic wells of multiwell plates), as well as cells directly attached to a glass substrate such as the glass of a vial.
This means that a laboratory can obtain frozen cell cultures in a desired format for testing, maintain the cultures in the freezer until they are needed, and then inoculate the thawed cultures as needed. Thus, the laboratory does not have to have the capability and funds available to maintain a cell culture service. For example, cells in the ready-to-use format of the present invention that are useful in the diagnosis of viral infections (e.g., respiratory viruses, herpes, etc.) may be removed from the freezer, thawed, inoculated, and the answer regarding the presence or absence of virus determined within a matter of hours or a few days. In addition, these frozen cells can be used to detect other intracellular parasites (e.g., Chlamydia), as well as the presence of microbial toxins (e.g., toxins produced by various species of Clostridium).
By providing cell cultures in a ready-to-use format, the need for the operator (i.e., the microbiologist or technologist) to transfer cells from growth flasks to slides, multiwell plates, or shell vials is avoided. In addition, the present invention avoids the necessity for using cell culture hoods and facilities. Indeed, the use of shell vials in particularly preferred embodiments minimizes the need for advanced training in virological methods, as well as the need for special incubators (e.g., CO2 incubators commonly used with cell cultures), and space required to perform the tests. Shell vial inoculation, incubation, staining, and evaluation are relatively easy (See e.g., D. Leland, xe2x80x9cModified Virus Isolation Systems,xe2x80x9d in Clinical Virology, W. B. Saunders Co., Philadelphia, Pa. [1996], at pages 79-90). Although the vials require a special centrifuge carrier and a device to readily remove the coverelips from the bottom of the shell vials, the relative costs and inconvenience are small, as compared to the requirements of a typical cell culture laboratory. In addition, the use of shell vials can significantly shorten the times needed to make a diagnosis, and reduces the chances of contamination and/or deterioration of the cells, as compared to traditional cell culture methods. Thus, the present invention makes virology accessible to laboratories that are not equipped nor experienced with cell cultures.
The terms xe2x80x9csamplexe2x80x9d and xe2x80x9cspecimenxe2x80x9d in the present specification and claims are used in their broadest sense. On the one hand, they are meant to include a specimen or culture. On the other hand, they are meant to include both biological and environmental samples. These terms encompasses all types of samples obtained from humans and other animals, including but not limited to, body fluids such as urine, blood, fecal matter, cerebrospinal fluid (CSF), semen, sputum, and saliva, as well as solid tissue. These terms also refers to swabs and other sampling devices which are commonly used to obtain samples for culture of microorganisms.
Biological samples may be animal, including human, fluid or tissue, food products and ingredients such as dairy items, vegetables, meat and meat by-products, and waste. Environmental samples include environmental material such as surface matter, soil, water, and industrial samples, as well as samples obtained from food and dairy processing instruments, apparatus, equipment, disposable, and non-disposable items. These examples are not to be construed as limiting the sample types applicable to the present invention.
Whether biological or environmental, a sample suspected of containing microorganisms may (or may not) first be subjected to an enrichment means to create a xe2x80x9cpure culturexe2x80x9d of microorganisms. By xe2x80x9cenrichment meansxe2x80x9d or xe2x80x9cenrichment treatment,xe2x80x9d the present invention contemplates (i) conventional techniques for isolating a particular microorganism of interest away from other microorganisms by means of any culture medium and/or technique, and (ii) novel techniques for isolating particular microorganisms away from other microorganisms. It is not intended that the present invention be limited only to one enrichment step or type of enrichment means. For example, it is within the scope of the present invention, following subjecting a sample to a conventional enrichment means, to subject the resultant preparation to further purification such that a pure culture of a strain of a species of interest is produced. This pure culture may then be analyzed by the medium and method of the present invention.
As used herein, the term xe2x80x9corganismxe2x80x9d and xe2x80x9cmicroorganism,xe2x80x9d are used to refer to any species or type of microorganism, including but not limited to viruses and bacteria, including rickettsia and chlamydia. Thus, the term encompasses, but is not limited to DNA and RNA viruses, as well as organisms within the orders Rickettsiales and Chlamydiales.
As used herein, the term xe2x80x9cculture,xe2x80x9d refers to any sample or specimen which is suspected of containing one or more microorganisms. xe2x80x9cPure culturesxe2x80x9d are cultures in which the organisms present are only of one strain of a particular genus and species. This is in contrast to xe2x80x9cmixed cultures,xe2x80x9d which are cultures in which more than one genus and/or species of microorganism are present.
As used herein, the term xe2x80x9ccell type,xe2x80x9d refers to any ceil, regardless of its source or characteristics.
As used herein, the term xe2x80x9ccell line,xe2x80x9d refers to cells that are cultured in vitro, including primary cell lines, finite cell lines, continuous cell lines, and transformed cell lines.
As used herein, the terms xe2x80x9cprimary cell culture,xe2x80x9d and xe2x80x9cprimary culture,xe2x80x9d refer to cell cultures that have been directly obtained from animal or insect tissue. These cultures may be derived from adults as well as fetal tissue.
As used herein, the term xe2x80x9cfinite cell lines,xe2x80x9d refer to cell cultures that are capable of a limited number of population doublings prior to senescence.
As used herein, the term xe2x80x9ccontinuous cell lines,xe2x80x9d refer to cell cultures that have undergone a xe2x80x9ccrisisxe2x80x9d phase during which a population of cells in a primary or finite cell line apparently ceases to grow, but yet a population of cells emerges with the general characteristics of a reduced cell size, higher growth rate, higher cloning efficiency, increased tumorigenicity, and a variable chromosomal complement. These cells often result from spontaneous transformation in vitro. Thuse cells have an indefinite lifespan.
As used herein, the term xe2x80x9ctransformed cell lines,xe2x80x9d refers to cell cultures that have been transformed into continuous cell lines with the characteristics as described above. Transformed cell lines can be derived directly from tumor tissue and also by in vitro transformation of cells with whole virus (e.g., SV40 or EBV), or DNA fragments derived from a transforming virus using vector systems.
As used herein, the term xe2x80x9chybridomas,xe2x80x9d refers to cells produced by fusing two cell types together. Commonly used hybridomas include those created by the fusion of antibody-secreting B cells from an immunized animal, with a malignant myeloma cell line capable of indefinite growth in vitro. These cells are cloned and used to prepare monoclonal antibodies.
As used herein, the term xe2x80x9cmixed cell culture,xe2x80x9d refers to a mixture of two types of cells. In some preferred embodiments, the cells are cell lines that are not genetically engineered, while in other preferred embodiments the cells are genetically engineered cell lines. In some embodiments, the one or more of the cell types is re xe2x80x9cpermissivexe2x80x9d (i.e., virus is capable of replication and spread from cell to cell within the culture). The present invention encompasses any combination of cell types suitable for the detection, identification, and/or quantitation of viruses in samples, including mixed cell cultures in which all of the cell types used are not genetically engineered, mixtures in which one or more of the cell types are genetically engineered and the remaining cell types are not genetically engineered, and mixtures in which all of the cell types are genetically engineered.
As used herein, the term xe2x80x9csuitable for the detection of intracellular parasites,xe2x80x9d refers to cell cultures that can be successfully used to detect the presence of an intracellular parasite in a sample. In preferred embodiments, the cell cultures are capable of maintaining their susceptibility to infection and/or support replication of the intracellular parasite. It is not intended that the present invention be limited to a particular cell type or intracellular parasite.
As used herein, the term xe2x80x9csusceptible to infectionxe2x80x9d refers to the ability of a cell to become infected with virus or another intracellular organism. Although it encompasses xe2x80x9cpermissivexe2x80x9d infections, it is not intended that the term be so limited, as it is intended that the term encompass circumstances in which a cell is infected, but the organism does not necessarily replicate and/or spread from the infected cell to other cells. The phrase xe2x80x9cviral proliferation,xe2x80x9d as used herein describes the spread or passage of infectious virus from a permissive cell type to additional cells of either a permissive or susceptible character.
As used herein, the term xe2x80x9ctoxinxe2x80x9d refers to any substance (usually a protein or conjugated protein) that is detrimental (i.e., poisonous) to cells and/or organisms. In particularly preferred embodiments, the term refers to extracellular toxins produced by various bacterial species, including, but not limited to the members of the genus Clostridium. However, it is not intended that the present invention be limited to any particular toxin or bacterial species. Indeed, it is intended that the term encompass toxins produced by any organism.
As used herein, the terms xe2x80x9cmonolayer,xe2x80x9d xe2x80x9cmonolayer culture,xe2x80x9d and xe2x80x9cmonolayer cell culture,xe2x80x9d refer to cells that have adhered to a substrate and grow in as a layer that is one cell in thickness. Monolayers may be grown in any format, including but not limited to flasks, tubes, coverslips, wells of microtiter plates, roller bottles, etc. Cells may also be grown attached to microcarriers, including but not limited to beads. In particularly preferred embodiments, the monolayers of the present invention are grown on coverslips placed within shell vials.
As used herein, the term xe2x80x9csuspension,xe2x80x9d and xe2x80x9csuspension culture,xe2x80x9d refers to cells that survive and proliferate without being attached to a substrate. Suspension cultures are typically produced using hematopoietic cells, transformed cell lines, and cells from malignant tumors.
As used herein, the terms xe2x80x9cculture media,xe2x80x9d and xe2x80x9ccell culture media,xe2x80x9d refers to media that are suitable to support the growth of cells in vitro (i.e., cell cultures). It is not intended that the term be limited to any particular culture medium. For example, it is intended that the definition encompass outgrowth as well as maintenance media. Indeed, it is intended that the term encompass any culture medium suitable for the growth of the cell cultures of interest.
As used herein, the term xe2x80x9cobligate intracellular parasitexe2x80x9d (or xe2x80x9cobligate intracellular organism) refers to any organism which requires an intracellular environment for its survival and/or replication. Obligate intracellular parasites include viruses, as well as many other organisms, including certain bacteria (e.g., most members of the orders Rickettsiales [e.g., Coxiella, Rickettsia and Ehrlichia] and Chlamydiales [e.g., C. trachomatis, C. psittaci], etc). The term xe2x80x9cintracellular parasite,xe2x80x9d refers to any organism that may be found within the cells of a host animal, including but not limited to obligate intracellular parasites briefly described above. For example, intracellular parasites include organisms such as Brucella, Listeria, Mycobacterium (e.g., M. tuberculosis and M. leprae), and Plasmodium, as well as Rochalimea.
As used herein, the term xe2x80x9cantimicrobial,xe2x80x9d is used in reference to any compound which inhibits the growth of, or kills microorganisms. It is intended that the term be used in its broadest sense, and includes, but is not limited to compounds such as antibiotics which are produced naturally or synthetically. It is also intended that the term includes compounds and elements that are useful for inhibiting the growth of, or killing microorganisms.
The terms xe2x80x9cconfluentxe2x80x9d or xe2x80x9cconfluencyxe2x80x9d as used herein in reference to an adherent cell line define a condition wherein cells throughout a culture are in contact with each other creating what appears to be a continuous sheet or xe2x80x9cmonolayerxe2x80x9d of cells.
The terms xe2x80x9ccytopathic effectxe2x80x9d or xe2x80x9cCPExe2x80x9d as used herein describe changes in cellular structure (i.e., a pathologic effect) resulting from external agents such viruses and/or toxins. Common cytopathic effects include cell destruction, syncytia (i.e., fused giant cells) formation, cell rounding vacuole formation, and formation of inclusion bodies. CPE results from actions of a virus on permissive cells that negatively affect the ability of the permissive cellular host to preform its required functions to remain viable. In in vitro cell culture systems, CPE is evident when cells, as part of a confluent monolayer, show regions of non-confluence after contact with a specimen that contains a virus. The observed microscopic effect is generally focal in nature and the foci is initiated by a single virion. However, depending upon viral load in the sample, CPE may be observed throughout the monolayer after a sufficient period of incubation. Cells demonstrating viral induced CPE usually change morphology to a rounded shape, and over a prolonged period of time can die and be released form their anchorage points in the monolayer. When many cells reach the point of focal destruction, the area is called a viral plaque, which appears as a hole in the monolayer. Cytopathic effects are readily discernable and distinguishable by those skilled in the art.
As used herein, the term xe2x80x9csubstratexe2x80x9d refers to a physical support to which cells are capable of binding so as to produce a monolayer. In some embodiments, plastics are used as substrates for production and maintenance of monolayers, while in other preferred embodiments, glass is used as the substrate. However, it is not intended that the present invention be limited to any particular substrate.
As used herein, the term xe2x80x9cglassxe2x80x9d refers to the commonly used material. In particular, the term refers to hard, brittle, often transparent or translucent materials that are produced by fusing silicates with soda or potash, lime, and in some cases, various metallic oxides. In particularly preferred embodiments of the invention, type 2 glass is used. However, it is not intended that the present invention be limited to any particular type or formula of glass.
The term xe2x80x9cmultiwellxe2x80x9d refers to any device that has multiple wells. In particularly preferred embodiments of the present invention plastic multiwell plates are used. These multiwell plates include, but are not limited to the two, four, eight, 16, 24, and 48 well plates commonly used in cell culturing methods, as well as microtiter plates (e.g., 96-well plates), and other suitable plate formats. It is not intended that the present invention be limited to any particular plate or number of wells. Indeed, it is contemplated that various multiwell plates will find use in the present invention.
The abbreviation xe2x80x9cONPG,xe2x80x9d represents o-Nitrophenyl-xcex2-D-Galactopyranoside. ONPG is a substrate for the enzyine xcex2-galactosidase (xcex2-gal). The reaction between ONPG and xcex2-gal produces a yellow product which can be quantified spectrophotometrically at 405 nm.
The abbreviation xe2x80x9cX-gal,xe2x80x9d represents the chemical compound 5-bromo-4-chloro-3-indolyl-xcex2-D-galactopyranoside, a substrate for the enzyme xcex2-galactosidase. The reaction between X-gal and xcex2-galactosidase results in the formation of a blue precipitate which is visually discemable.
The term xe2x80x9chybriwix,xe2x80x9d represents a product of Diagnostic Hybrids, Inc., Athens, Ohio which allows for quantification of certain viral DNA in an infected monolayer of cells by DNA hybridization. xe2x80x9cDNA hybridizationxe2x80x9d is the annealing of two complementary DNA molecules whose base sequences match according to the rules of base pairing. DNA hybridization is used to identify or quantify an unknown or xe2x80x9ctargetxe2x80x9d DNA by hybridization to a known DNA or xe2x80x9cprobe.xe2x80x9d The probe is typically labeled with a reporter molecule such as 125I, a radioisotope which can be detected and quantified with a gamma counter.
The phrase xe2x80x9cplaque reduction assay,xe2x80x9d or xe2x80x9cPRA,xe2x80x9d as used herein describes a standard method used to determine efficacy of anti-viral drugs by enumerating a decrease in plaque formation in a cell monolayer exposed to a drug. A xe2x80x9cplaquexe2x80x9d is a defined area of xe2x80x9cCPE.xe2x80x9d It is usually the result of infection of the cell monolayer with a single infectious virus which then replicates and spreads to adjacent cells of the monolayer. A plaque may also be referred to as a xe2x80x9cfocus of viral infection.xe2x80x9d
The term xe2x80x9cpermissivexe2x80x9d as used herein describes the sequence of interactive events between a virus and its putative host cell. The process begins with viral adsorption to the host cell surface and ends with release of infectious virions. A cell is xe2x80x9cpermissivexe2x80x9d if it readily permits the spread of virus to other cells. Many methods are available for the determination of the permissiveness of a given cell line, including, but not limited to plaque reduction assays, comparisons of the production and/or quantitation of viral proteins based on results obtained from gel electrophoresis, relative comparisons using hybridization analysis to analyze DNA or RNA content, etc.
The term xe2x80x9csusceptible,xe2x80x9d as used herein describes the extent that a permissive or non-permissive host cell can adsorb and be penetrated by a virus. A cell line may be susceptible without being permissive in that it can be penetrated but not release virions. A permissive cell line however must be susceptible.
The phrase xe2x80x9cseed on,xe2x80x9d as used herein describes the act of transferring an aqueous solution of suspended cells into a vessel containing cells adhered to a surface, after which the vessel is stored for a sufficient period of time to allow the suspended cells or xe2x80x9cseedsxe2x80x9d to settle out by gravity and attach in a relatively uniform manner to the adhered cells and become integrated into the final cell monolayer as a mixture. A xe2x80x9cmixed cell monolayer,xe2x80x9d results from the xe2x80x9cseed onxe2x80x9d process.
The phrase xe2x80x9cseed in,xe2x80x9d as used herein describes the mixing of two or more aqueous solutions of suspended tissue culture cells, each cell suspension having different cellular properties, and transfer of such mixture of cells into a vessel which is stored for a sufficient period of time to allow the suspended cells to settle out by gravity and attach in a relatively uniform manner such that the distribution of any single cell type is indicative of the relative ratio of the cells in the original mixture.
As used herein, the terms xe2x80x9cchromogenic compound,xe2x80x9d and xe2x80x9cchromogenic substrate,xe2x80x9d refer to any compound useful in detection systems by their light absorption or emission characteristics. The term is intended to encompass any enzymatic cleavage products, soluble, as well as insoluble, which are detectable either visually or with optical machinery. Included within the designation xe2x80x9cchromogenicxe2x80x9d are all enzymatic substrates which produce an end product which is detectable as a color change. This includes, but is not limited to any color, as used in the traditional sense of xe2x80x9ccolors,xe2x80x9d such as indigo, blue, red, yellow, green, orange, brown, etc., as well as fluorochromic or fluorogenic compounds, which produce colors detectable with fluorescence (e.g., the yellow-green of fluorescein, the red of rhodamine, etc.). It is intended that such other indicators as dyes (e.g., pH) and luminogenic compounds be encompassed within this definition.
As used herein, the commonly used meaning of the terms xe2x80x9cpH indicator,xe2x80x9d xe2x80x9credox indicator,xe2x80x9d and xe2x80x9coxidation-reduction indicator,xe2x80x9d are intended. Thus, xe2x80x9cpH indicator,xe2x80x9d encompasses all compounds commonly used for detection of pH changes, including, but not limited to phenol red, neutral red, bromthymol blue, bromcresol purple, bromoresol green, bromchlorophenol blue, m-cresol purple, thymol blue, bromcresol purple, xylenol blue, methyl red, methyl orange, and cresol red. The terms xe2x80x9credox indicator,xe2x80x9d and xe2x80x9coxidation-reduction indicator,xe2x80x9d encompasses all compounds commonly used for detection of oxidation/reduction potentials (i.e., xe2x80x9ceHxe2x80x9d) including, but not limited to various types or forms of tetrazolium, resazurin, and methylene blue.
As used herein, the term xe2x80x9cinoculating suspension,xe2x80x9d or xe2x80x9cinoculant,xe2x80x9d is used in reference to a suspension which may be inoculated with organisms to be tested. It is not intended that the term xe2x80x9cinoculating suspension,xe2x80x9d be limited to a particular fluid or liquid substance. For example, inoculating suspensions may be comprised of water, saline, or an aqueous solution. It is also contemplated that an inoculating suspension may include a component to which water, saline or any aqueous material is added. It is contemplated in one embodiment, that the component comprises at least one component useful for the intended microorganism. It is not intended that the present invention be limited to a particular component.
As used herein, the term xe2x80x9ckit,xe2x80x9d is used in reference to a combination of reagents and other materials.
As used herein, the term xe2x80x9cprimary isolation,xe2x80x9d refers to the process of culturing organisms directly from a sample. As used herein, the term xe2x80x9cisolation,xe2x80x9d refers to any cultivation of organisms, whether it be primary isolation or any subsequent cultivation, including xe2x80x9cpassage,xe2x80x9d or xe2x80x9ctransfer,xe2x80x9d of stock cultures of organisms for maintenance and/or use.
As used herein, the term xe2x80x9cpresumptive diagnosis,xe2x80x9d refers to a preliminary diagnosis which gives some guidance to the treating physician as to the etiologic organism involved in the patient""s disease. Presumptive diagnoses are often based on xe2x80x9cpresumptive identifications,xe2x80x9d which as used herein refer to the preliminary identification of a microorganism.
As used herein, the term xe2x80x9cdefinitive diagnosis,xe2x80x9d is used to refer to a final diagnosis in which the etiologic agent of the patient""s disease has been identified. The term xe2x80x9cdefinitive identificationxe2x80x9d is used in reference to the final identification of an organism to the genus and/or species level.
The term xe2x80x9crecombinant DNA molecule,xe2x80x9d as used herein refers to a DNA molecule which is comprised of segments of DNA joined together by means of molecular biological techniques.
The terms xe2x80x9creporter gene construct,xe2x80x9d or xe2x80x9creporter gene vector,xe2x80x9d as used herein refers to a recombinant DNA molecule containing a sequence encoding the product of a reporter gene and appropriate nucleic acid sequences necessary for the expression of the operably linked coding sequence in a particular host organism. Eukaryotic cells are known to utilize promoters, enhancers, and termination and polyadenylation signals.
The term xe2x80x9creporter gene,xe2x80x9d refers to an oligonucleotide having a sequence encoding a gene product (typically an enzyme) which is easily and quantifiably assayed when a construct comprising the reporter gene sequence operably linked to a heterologous promoter and/or enhancer element is introduced into cells containing (or which can be made to contain) the factors necessary for the activation of the promoter and/or enhancer elements. Examples of reporter genes include but are not limited to bacterial genes encoding xcex2-galactosidase (lacZ), the bacterial chloramphenicol acetyltransferase (cat) genes, firefly luciferase genes and genes encoding xcex2-glucuronidase (GUS).
The term xe2x80x9cgenetically engineered cell line,xe2x80x9d refers to a cell line that contains heterologous DNA introduced into the cell line by means of molecular biological techniques (i.e., recombinant DNA technology).
The term xe2x80x9cstable transfection,xe2x80x9d or xe2x80x9cstably transfected,xe2x80x9d refers to the introduction and integration of foreign DNA into the genome of the transfected cell. The term xe2x80x9cstable transfectant,xe2x80x9d refers to a cell which has stably integrated foreign DNA into the genomic DNA.
The term xe2x80x9cstable transfectionxe2x80x9d (or xe2x80x9cstably transfectedxe2x80x9d), refers to the introduction and integration of foreign DNA into the genome of the transfected cell. The term xe2x80x9cstable transfectant,xe2x80x9d refers to a cell which has stably integrated foreign DNA into the genomic DNA.
The term xe2x80x9cselectable marker,xe2x80x9d as used herein refers to the use of a gene which encodes an enzymatic activity that confers resistance to an antibiotic or drug upon the cell in which the selectable marker is expressed. Selectable markers may be xe2x80x9cdominantxe2x80x9d; a dominant selectable marker encodes an enzymatic activity which can be detected in any mammalian cell line. Examples of dominant selectable markers include the bacterial aminoglycoside 3xe2x80x2 phosphotransferase gene (also referred to as the neo gene) which confers resistance to the drug G418 in mammalian cells, the bacterial hygromycin G phosphotransferase (hyg) gene which confers resistance to the antibiotic hygromycin and the bacterial xanthine-guanine phosphoribosyl transferase gene (also referred to as the gpt gene) which confers the ability to grow in the presence of mycophenolic acid. Other selectable markers are not dominant in that their use must be in conjunction with a cell line that lacks the relevant enzyme activity. Examples of non-dominant selectable markers include the thymidine kinase (tk) gene which is used in conjunction with tkxe2x88x92cell lines, the CAD gene which is used in conjunction with CAD-deficient cells and the mammalian hypoxanthine-guanine phosphoribosyl transferase (hprt) gene which is used in conjunction with hprtxe2x88x92cell lines. A review of the use of selectable markers in mammalian cell lines is provided in Sambrook et al. (Sambrook et al., supra at pp.16.9-16.15).
The following examples are provided in order to demonstrate and further illustrate certain preferred embodiments and aspects of the present invention and are not to be construed as limiting the scope thereof.
In the experimental disclosure which follows, the following abbreviations apply: eq (equivalents); M (Molar); xcexcM (micromolar); N (Normal); mol (moles); mmol (millimoles); xcexcmol (micromoles); nmol (nanomoles); g (grams); mg (milligrams); xcexcg (micrograms); ng (nanograms); pg (picograms); l or L (liters); ml (milliliters); xcexcl (microliters); cm (centimeters); mm (millimeters); xcexcm (micrometers); nm (nanometers); xg (times gravity);xc2x0 C. (degrees Centigrade); FBS (fetal bovine serum); PBS (phosphate buffered saline; HEPES (N-[2-hydroxyethyl]piperazine-Nxe2x80x2-[2-ethanesulfonic acid]); HBSS (Hank""s Balanced Salt Solution); MEM (Minimal Essential Medium); EMEM (Eagle""s Minimal Essential Medium); DMSO (dimethyl sulfoxide); ELVIS(copyright) RM (ELVIS(copyright) Replacement Medium); PFU (plaque forming unit); TNTC (too numerous to count); FITC (fluorescein isothiocyanate); Lee Laboratories (Lee Laboratories, Grayson, Ga.); ELVIS(copyright) (enzyme-linked virus inducible system) HSV cells; CDT (Clostridium difficile toxin); Diagnostic Hybrids (Diagnostic Hybrids, Inc., Athens, Ohio); Promega (Promega, Corporation, Madison, Wis.); Kimble (Kimble/Kontes, Vineland, N.J.); Bellco (Bellco Glass Inc., Vineland, N.J.); Costar (Acton, Mass.); Falcon (Franklin Lakes, N.J.); Chemicon (Chemicon, Inc., Temecula, Calif.); BBL (Becton Dickinson Microbiology Systems, Cockeysville, Md.); DIFCO (Difco Laboratories, Detroit, Mich.); U.S. Biochemical (U.S. Biochemical Corp., Cleveland, Ohio); Chemicon (Chemicon, Inc., Temecula, Calif.); Fisher (Fisher Scientific, Pittsburgh, Pa.); Sigma (Sigma Chemical Co., St. Louis, Mo.); ATCC (American Type Culture Collection, Rockville, Md.); Bartel""s (Bartel""s, Issaquah, Wash.); and BioWhittaker (BioWhittaker, Walkersville, Md.).